The present invention relates to nucleic acid and amino acid sequences of human T-type calcium channel variants and the use of these sequences in diagnosis of disease states associated with pain and for use as targets for screening therapeutic compounds useful in the treatment of disease states associated with pain.
Voltage-gated calcium channels can be divided into high- and low-threshold types. The high-threshold channels include the dihydropyridine-sensitive L-type, the xcfx89-conotoxin GVIA-sensitive N-type and xcfx89-agatoxin IVA-sensitive P-type. Depending on the tissue, these channel subtypes consist of xcex11, xcex12xcex4, xcex2 and xcex3 subunits. (Perez-Reyes and Schneider (1995) Kid. Int. 48:1111-1124.) To date, only one type of low-threshold calcium channel is known, the T-type calcium channel.
T-type calcium channels have hyperpolarized steady-state inactivation characteristics, a low threshold for inactivation, small single channel conductance and display rapid inactivation kinetics. (Ertel and Ertel (1997) Trends Pharmacol. Sci. 18:37-42.) The functional roles for T-type calcium channels in neurons include membrane depolarization, calcium entry and burst firing. (White et al. (1989) Proc. Natl. Acad. Sci. USA 86:6802-6806.) T-type calcium channels are found in many neurons of the central and peripheral nervous systems, including small and medium diameter neurons of the dorsal root ganglia (Scroggs and Fox (1992) J. Physiol. 445:639-658) and neurons in the thalamus. (Suzuki and Rogawski (1989) Proc. Natl. Acad. Sci. USA 86:7228-7232.)
Calcium currents have been found to be important in several neurological and muscular functions, e.g., pain transmission, cardiac pacemaker activity, etc. Improper functioning of these channels has been implicated in arrythmias, chronic peripheral pain, improper pain transmission in the central nervous system, and epilepsy.
Anti-epileptic drugs are known to cause a reduction of the low-threshold calcium current (LTCC or T-type Ca2+ current) in thalamic neurons. (Coulter et al.(1989) Ann. Neurol. 25:582-593.) One such anti-epileptic compound, ethosuximide, has been shown to fully block T-type Ca2+ current in freshly dissected neurons from dorsal root ganglia (DRG neurons) of adult rats (Todorovic and Lingle (1998) J. Neurophysiol. 79:240-252), and may have limited efficacy in the treatment of abnormal, chronic pain syndromes that follow peripheral nerve damage.
Molecular cloning has revealed the cDNA and corresponding amino acid sequences of several different xcex11 subunits (xcex11A, xcex11B, xcex11C, xcex11D, xcex11E, xcex11G, xcex11H, xcex11I, and xcex11S). While the cloned xcex11 subunits identified thus far correspond to several of the calcium channels found in cells, they do not account for all types of calcium conductance found in native cells.
The present invention relates to the discovery of human T-type calcium channel xcex11I subunit variants that are useful in diagnosis of disease states associated with the peripheral nervous system and for screening compounds that may be used in the treatment of mammals for these disease states.
The invention is based on the discovery of human T-type calcium channel xcex11I subunit variants (TCCV-1 and TCCV-2), the polynucleotides encoding TCCV-1 or TCCV-2, and the use of these compositions in screening for compounds effective in treating disease states associated with peripheral pain, and the use of these compositions for diagnosis of these disease states. In particular, the present invention expression vectors, host cells, antibodies, diagnostic kits, and transgenic/knockout animals are provided.
The invention features an isolated polynucleotide encoding TCCV-1 or TCCV-2 polypeptides. The invention further provides an isolated polynucleotide, encoding a TCCV-1 or TCCV-2 polypeptide wherein the polynucleotide encodes an TCCV-1 or TCCV-2 polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 4, respectively. In certain embodiments, the polynucleotide is detectably labeled or is complementary to the polynucleotide encoding a TCCV-1 or TCCV-2 polypeptide. The complementary polynucleotide can also be detectably labeled. In another embodiment, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:1 or 3.
The present invention encompasses an expression vector comprising the polynucleotide encoding SEQ ID NO:2 or 4. Also contemplated is a host cell comprising the polynucleotide encoding SEQ ID NO:2 or 4. The host cell can be a prokaryotic or eukaryotic cell. The invention further comprises a method of producing a TCCV-1 or TCCV-2 polypeptide comprising: culturing the host cell comprising the expression vector comprising the polynucleotide encoding SEQ ID NO:2 or 4 under conditions suitable for expression of the polypeptide; and recovering the polypeptide from the host cell.
The present invention also contemplates a method of detecting a polynucleotide encoding a TCCV-1 or TCCV-2 polypeptide in a sample containing nucleic acid material, comprising the steps of: contacting the sample with a polynucleotide which is the complement of the polynucleotide encoding SEQ ID NO:2 or 4, wherein the complement is detectably labeled, under conditions suitable for formation of a hybridization complex; and detecting the complex, wherein the presence of the complex is indicative of the presence of the polynucleotide encoding the polypeptide in the sample.
The present invention provides a diagnostic test kit comprising: the polynucleotide comprising SEQ ID NO:1 or 3; and instructions for conducting the diagnostic test.
The present invention encompasses a method of screening for a compound that modulates TCCV-1 or TCCV-2 activity comprising: contacting TCCV-1 or TCCV-2, or fragment thereof with the compound; and detecting modulation of TCCV-1 or TCCV-2 activity. In certain embodiments, the TCCV-1 or TCCV-2 is expressed on a cell or tissue or immobilized on a solid support. The compound can be an antagonist or agonist of TCCV-1 or TCCV-2 activity. In a further embodiment, the compound is ethosuximide or an analog thereof.
The present invention provides an isolated TCCV-1 or TCCV-2 polypeptide or fragment thereof. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:2 or 4. The polypeptide is recombinantly produced or synthetically produced. The present invention also provides an isolated antibody which specifically binds to the polypeptide of SEQ ID NO:2 or 4.
The present invention encompasses a transgenic nonhuman mammal comprising the polynucleotide encoding TCCV-1 or TCCV-2 polypeptide. The transgenic nonhuman mammal can also comprise the polynucleotide which is the complement of the polynucleotide encoding TCCV-1 or TCCV-2 which is capable of hybridizing to a polynucleotide encoding TCCV-1 or TCCV-2, thereby reducing expression of TCCV-1 or TCCV-2.